Research ArticleResveratrol Prevents Right Ventricle Remodeling andDysfunction in Monocrotaline-Induced Pulmonary ArterialHypertension with a Limited Improvement in theLung Vasculature
Eduardo Vázquez-Garza,1 Judith Bernal-Ramírez,1 Carlos Jerjes-Sánchez,1,2
Omar Lozano,1,2 Edgar Acuña-Morín,1 Mariana Vanoye-Tamez,1
Martín R. Ramos-González,1 Héctor Chapoy-Villanueva,1 Luis Pérez-Plata,1
Luis Sánchez-Trujillo,1,3 Guillermo Torre-Amione,1,4 Alicia Ramírez-Rivera,5
and Gerardo García-Rivas 1,2
1Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Ave. Morones Prieto 3000, 64710 Monterrey, N.L., Mexico2Centro de Investigación Biomédica, Hospital Zambrano-Hellion, Tec Salud, Tecnologico de Monterrey, Batallón San Patricio 112Col. Real de San Agustín, 66278 San Pedro Garza García, N.L., Mexico3Unidad de Investigación Clínica en Medicina SC, 64718 Monterrey, N.L., Mexico4Weill Cornell Medical College, Methodist DeBakey Heart & Vascular Center, The Methodist Hospital, Houston, 77030 TX, USA5Unidad de Investigación Clínica en Medicina, 64718 Monterrey, N.L., Mexico
Correspondence should be addressed to Gerardo García-Rivas; [email protected]
Received 21 September 2019; Revised 24 December 2019; Accepted 7 January 2020; Published 4 February 2020
Pulmonary arterial hypertension (PAH) is a life-threatening disease that is characterized by an increase in pulmonary vascularpressure, leading to ventricular failure and high morbidity and mortality. Resveratrol, a phenolic compound and a sirtuin 1pathway activator, has known dietary benefits and is used as a treatment for anti-inflammatory and cardiovascular diseases. Itstherapeutic effects have been published in the scientific literature; however, its benefits in PAH are yet to be precisely elucidated.Using a murine model of PAH induced by monocrotaline, the macroscopic and microscopic effects of a daily oral dose ofresveratrol in rats with PAH were evaluated by determining its impact on the lungs and the right and left ventricular function.While most literature has focused on smooth muscle cell mechanisms and lung pathology, our results highlight the relevance oftherapy-mediated improvement of right ventricle and isolated cardiomyocyte physiology in both ventricles. Although significantdifferences in the pulmonary architecture were not identified either micro- or macroscopically, the effects of resveratrol on rightventricular function and remodeling were observed to be beneficial. The values for the volume, diameter, and contractility of theright ventricular cardiomyocytes returned to those of the control group, suggesting that resveratrol has a protective effect againstventricular dysfunction and pathological remodeling changes in PAH. The effect of resveratrol in the right ventricle delayed theprogression of findings associated with right heart failure and had a limited positive effect on the architecture of the lungs. Theuse of resveratrol could be considered a future potential adjunct therapy, especially when the challenges to making a diagnosisand the current therapy limitations for PAH are taken into consideration.
HindawiOxidative Medicine and Cellular LongevityVolume 2020, Article ID 1841527, 13 pageshttps://doi.org/10.1155/2020/1841527
Pulmonary arterial hypertension (PAH) is a rare but progres-sive and often fatal pulmonary vascular disease [1]. PAH ischaracterized by a progressive increase in pulmonary vascu-lar resistance and pulmonary arterial pressure, with second-ary vascular and right ventricular (RV) remodeling, RVdysfunction, heart failure syndromes, and, finally, prematuredeath [2]. Currently, approved therapies target three mainpathways important in endothelial function: the prostacyclinand nitric oxide pathways, which are underexpressed, andthe endothelin pathway, which is overexpressed in PAHpatients [3]. PAH triggers a series of events on RV function,including activation of several signaling pathways thatregulate cell growth,metabolism, extracellular matrix remodel-ing, and energy production [4, 5]. Right heart failure syndromeemerges in the setting of ischemia, alterations in substrateand mitochondrial energy metabolism, increased free oxygenradicals, increased cell loss, downregulation of adrenergicreceptors, increased inflammation and fibrosis, and patho-logic microRNA expression [4]. Current therapeutic schemeshave not been able to regulate these mechanisms in the longterm; therefore, there is a need for more successful strategiesto manage right ventricular heart failure in the future [4].
Although the current treatment improves quality of lifeand survival [6, 7], a therapeutic approach to improve theRV function is lacking. Resveratrol (RES) is a phenoliccompound with a known cardioprotective effect in severalcardiovascular diseases [8]. However, its primary mecha-nisms of action have yet to be fully elucidated but includesirtuin modulation, reactive oxygen species (ROS) scaveng-ing, and antioxidant mechanisms [9, 10]. The in vitro use ofRES has been demonstrated to reduce oxidative stress andincrease cell survival, inhibiting apoptosis and modulatingthe cell cycle in several cell lines [11]. RES has also beenreported to have antifibrotic and anti-inflammatory effectsin vivo [12]. This compound has been evaluated in somePAH animal models for its ability to decrease lung damagein the tissue or pulmonary trunk [13], but the molecularmechanism of cardioprotection afforded by RES remainsonly partially understood. Thus, in this study, the effect ofRES in a PAH model on the lungs and ventricles wasassessed in its ability to delay PAH progression. To achievethis, we performed an echocardiographic assessment toevaluate ventricular function, macroscopic and histologicchanges, as well as contractile modifications, and biomarkerexpression in isolated cells. RES was demonstrated to bepreferentially cardioprotective of the function and structureof the right ventricle, and it was shown to have a limitedeffect on the pulmonary vasculature.
2. Materials and Methods
2.1. Murine Model of Pulmonary Hypertension. All animalstudies were approved by the Internal Committee for Careand Handling of Laboratory Animals of the School of Medi-cine of the Tecnologico de Monterrey (Protocol no. 2017-006) and were performed following the Mexican NationalLaboratory Animal Health Guidelines (NOM 062-ZOO
1999). Experiments were performed on adult male Spra-gue–Dawley rats (Bioinvert, MX), weighing >300 g. Pulmo-nary hypertension was induced by a single subcutaneousinjection of monocrotaline (MC) (60mg/kg body weight)diluted in dimethylsulfoxide (DMSO, Sigma-Aldrich, St.Louis, MO, USA). DMSO was used with the same volumefor both groups of control rats and only RES rats. Animalswere kept in a controlled temperature environment with a12 h light cycle. Water and food were given ad libitum. Toassess the effect of RES (Trans-isomer, RyTLabs), we dividedthe specimens into four groups: control rats (CTRL, n = 12),monocrotaline-treated rats (PAH, n = 12), rats treated withMC and RES (20mg/kg per day, by gavage) (PAH+RES, n =11), and only RES rats (20mg/kg/day, by gavage) (RES, n =13), from day 1 to day 42 after injection. All animals wereobserved for general appearance and respiratory symptom-atology. Disease progression was characterized by anatomicalpostmortem data and echocardiography, which correlatestrongly with the right heart catheterization measurements.
2.2. Echocardiographic Assessment of Cardiac Function.Noninvasive, transthoracic cardiac ultrasonography was per-formed 35 days after MC/DMSO injection, with a PhilipsEnVisor Ultrasound (Philips Healthcare, Andover, MA)equipped with a 12MHz S-type transducer, under 1-3%sevoflurane anesthesia. After placing the animal on a thermalpad, with the chest shaved and using ultrasound transmis-sion gel, standard views recommended by the AmericanSociety of Echocardiography were obtained. A parasternalshort-axis view at the level of the great vessels was used tomeasure pulmonary artery flow, using pulse wave Dopplermode with a sample gate of 1.0mm just proximal to the pul-monary valve. Here, we measured the pulmonary arteryvelocity time integral (VTI), pulmonary ejection time (ET),peak pulmonary flow velocity, and the pulmonary arteryacceleration time (PAAT). A ratio between PAAT and ETwas obtained, and the mean pulmonary artery pressure(mPAP) was estimated using the formula mPAP = 58:7 −ð1:21 × PAATÞ. By combining pulmonary artery velocity-time integral, pulmonary artery area, and heart rate, echo-cardiographically derived cardiac output was determined,as previously published [14]. RV free wall thickness wasmeasured at end-diastole from the parasternal long-axis viewusing M-mode. The apical four-chamber view was employedto measure the end-diastolic RV diameter and M-mode-derived tricuspid annular plane systolic excursion (TAPSE).Left ventricle (LV) diameters and fractional shortening(FS = diastolic LVID − systolic LVID/diastolic LVID × 100[LVID (LV internal diameter)]) were measured with M-mode from the short-axis view at the level of the papillarymuscles. Subsequently, the echocardiographic RV/LV end-diastolic diameter ratio was calculated and used as an assess-ment of RV enlargement.
2.3. Histological Preparations. After 42 days of RES treat-ment, the specimens were euthanized. Hearts were quicklyexcised from the rats after being anesthetized with inhaled5% sevoflurane and sodium heparin (1000U/kg). The heartand lungs were dissected and weighed. The RV and LV were
2 Oxidative Medicine and Cellular Longevity
identified and isolated for different preparations. The sec-tions for histological findings were fixed in 4% (wt/vol)paraformaldehyde in PBS for at least 2 hours at roomtemperature, transferred to 70% ethanol, embedded in par-affin, and processed for hematoxylin/eosin (H&E) andMasson’s trichrome staining. Fibrotic index assessmentwas performed following previously published data [15]; inbrief, microphotographs were acquired using an Imager Z1Zeiss microscope with an AxioCam HRm and microphoto-graph processing with the AxioVision software. To assessfibrosis, we used a semiquantitative approach; after stainingwith Masson’s trichrome, we take microphotographs of thewhole slide at 2.5x, and the image is then decomposed in atleast seven fields at 5x. After the photos were taken, we quan-tified the number of blue and red pixels, and the results wererecorded to make a ratio of blue%/red% using ImageJ soft-ware. Data correspond to the analysis of 2 blinded analystsand three different fields. Cardiomyocyte area was assessedusing H&E slides. Microphotography of the papillary mus-cles was taken at 10x; only cells with a complete visible cyto-plasm and central nuclei were considered. At least ten cellsper photography were counted at two different levels. Allslides were analyzed using an object carrier with a capacityfor 7 slides, for their respective batches.
Regarding lung sections, the primary lung architecturewas assessed for each group using the H&E-stained slides.Predominant findings included inflammatory infiltrationand proliferation of the smooth muscular medial layer ofthe lung arterioles. We quantified the amount of these arteri-oles in seven random fields; vessels of an average 100μmwere selected to analyze diameter, luminal area, and occlu-sion. Occlusion was assessed by at least seven measurementsof the medial layer thickness for the average.
2.4. Cardiomyocyte Isolation. Ventricular myocytes were iso-lated following previously described methods [16]. Thehearts were excised and mounted on a Langendorff apparatusand perfused with Tyrode medium (TM), in mM: 128 NaCl,0.4 NaH2PO4, 6 glucose, 5.4 KCl, 0.5 MgCl-6H2O, 5 creati-nine, 5 taurine, and 25 HEPES, pH7.4 at 37°C, for 5minand digested by 0.1% collagenase type II (Worthington Bio-chemical, Lakewood, NJ) dissolved in TM. Afterwards, theRV and LV were dissected, and their cells mechanically dis-aggregated. Cardiomyocytes were rinsed with TM plus 0.1%albumin solution at increasing Ca2+ concentrations (0.25,0.5, and 1mM). Only rod-shaped cells were used in the stud-ies. All the confocal measurements were acquired using aLeica TCS SP5 confocal microscope equipped with a D-apochromatic 63x, 1.2NA, oil objective (Leica Microsystems,Wetzlar, Germany). To assess cell volume, freshly isolatedcardiomyocytes were incubated in TM with 5μM calcein-AM (Life Technologies, Carlsbad, CA, USA) at room temper-ature (RT) for 30min as previously described [17]. Then,cells were washed with a fluorophore-free and calcium-freesolution and images were taken at 400Hz, obtaining a stackof 2D images of 1μm section thickness every 1μm in the z-axis, covering the whole cell depth. A 488 nm wavelengthwas used to excite the fluorophore, and its emission was col-lected at 500-600 nm. Cell volume was evaluated as previ-
ously described [18]. Freshly isolated cardiomyocytes wereincubated in TM (1mM Ca2+) with 10μM Fluo-4AM (LifeTechnologies, Carlsbad, CA, USA) for 45min at RT. After-wards, the cells were washed with a fluorophore-free solu-tion, plated on laminin-covered glass coverslips andmounted in a superfusion chamber. Excitation and emissionwavelengths were 488nm and 500-600 nm, respectively. Cellshortening was evaluated under field stimulation (MYP100MyoPacer Field Stimulator; IonOptix, Milton, MA). The cellswere evaluated under field stimulation at 0.5, 1, and 2Hz, andline-scan images were recorded along the longitudinal axis ofthe cell at 400Hz with a one μm section thickness. Fluo-rescence data were normalized as ΔF/F0, where F is fluo-rescence intensity; all confocal microscopy images wereanalyzed using ImageJ.
2.5. Western Blotting. Total heart protein from right ven-tricles (30μg) was resolved on SDS-PAGE gel 15% andtransferred onto a PVDF membrane at 300mA for 2hours and incubated with anti-Acetylated-Lysine proteinantibody (9441S, Cell Signaling) (1 : 2000). The membranewas washed three times for 10min with PBS-0.5% Tween20 and subsequently probed with an HPR-conjugated sec-ondary antibody anti-rabbit IgG 1 : 5000 (sc-2004, SantaCruz) for 2 hours at room temperature. After washing threetimes for 10min, the blots were developed with SuperSignalWest Dura Extended Duration Substrate (Thermo Fisher Sci-entific, USA) and quantified by using a BioSpectrum 415Image Acquisition System (UVP, Upland, CA, USA). Anti-GAPDH antibody (1 : 2000) (ab9484, Abcam) was used as aloading control.
2.6.1. RNA Isolation, Reverse Transcription, and QuantitativePCR (qPCR). The total RNA from the tissue of the right ven-tricles was isolated using a TRIzol Reagent (15596026, Invi-trogen). The purity of all samples was confirmed measuringtheir 260/280 nm absorbance ratio using a Take3 multivol-ume plate in a Synergy HTmicroplate reader (BioTek Instru-ments). The cDNA was reverse-transcribed from 1μg of totalRNA using the SensiFAST cDNA Synthesis Kit (BIO-65053,Bioline). The qPCR reaction was performed using the Sensi-FAST SYBR Lo-ROX Kit (BIO-94020, Bioline) in a Quant-Studio 3 RT PCR System (Thermo Fisher Scientific) andthe data analyzed by the 2−ΔΔCt method to estimate eachgene’s mRNA expression. The primers were synthesized byT4 Oligo (Mexico). All primer sequences for BNP, collagen1, IL-1β, IL-10, troponin C, Sirt1, and HPRT as housekeep-ing genes are detailed in Supplementary Table 1.
2.6.2. Reagents. All chemical reagents were purchased fromSigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
2.7. Statistical Analysis. Statistical data are presented as themean ± SEM. Comparisons between means were made byunpaired Student’s t-test or one-way ANOVA followed byDunnett’s, Tukey’s, or Bonferroni’s post hoc tests whenappropriate to compare experimental groups. Differenceswere considered significant when p < 0:05. Data processing,
3Oxidative Medicine and Cellular Longevity
graphs, and statistical analysis were performed with Graph-Pad Prism (V.5.01; La Jolla, CA, USA).
3. Results
3.1. Resveratrol Had a Limited Effect on the Development of aMonocrotaline-Induced PAH Changes in the VascularArchitecture of the Lungs and the EchocardiographicPulmonary Artery Values. The study duration for this PAHmodel was 42 days as this was an adequate amount of timefor phenotypic changes (i.e., cyanosis in the extremities andweight loss) to take place. An increase in the weight of theheart and lungs was identified as a specific macroscopicchange. Compared to the untreated control group (CTRL)(1.4± 0.2 g), heart weight increased by 21% in the PAH group(1:7 ± 0:2 g) and 35% with 1:7 ± 0:3 g for the PAH+RESgroup. Lungs weight followed the same trend: a weightincrease of 2:0 ± 0:3 g was reported for the CTRL group,45% increase was observed for the PAH group (2:9 ± 0:3 g),and a 60% increase was seen for the PAH+RES group(3:2 ± 0:4 g). Normalized organ weight and the weight ofeach specimen showed the same trend (data not shown).There were no statistically significant changes in these valuesbetween the PAH+RES and PAH groups and no differencesbetween the CTRL group and the group treated only withRES. To exclude other cardiovascular pathologies, systemic,systolic, and diastolic blood pressure and heart rate valueswere evaluated. No differences were observed between thegroups (data not shown). The pathognomonic findings forlung vasculature in the PAH model included an increase inthe muscularized arteries, an increase in the lumen diameter,
and a concomitant decrease in luminal occlusion in the medialayer. The changes were due to the proliferation of the smoothmuscle cells and a slight increase in vessel diameter. Represen-tative microphotographs can be seen in Figure 1(a). RES wasunable to avoid the transformation of healthy vessels intomuscularized arteries; however, it diminished the amount ofthem (i.e., an 11.2-fold increase in the PAH+RES group anda 20-fold increase in the PAH group, representing a 56%decrease mediated by RES between these two groups), whencompared with its effect on the CTRL group (Figure 1(b)).There was a reduced effect of RES on the vascular lumendiameter (Figure 1(c)). Although there was a noticeableincrease of the PAH group compared to the CTRL group, thisvalue was not significantly decreased by RES, having 57:2 ±2:3 μm (a 1.59-fold increase in the PAH group and a 1.47-foldincrease in the PAH+RES group). This represented a 4%decrease in terms of the effect of RES on the PAH phenotype(i.e., 73:5 ± 2:4 μm vs. 70:7 ± 2:6 μm). Luminal occlusionfollowed a similar trend, with at least a 2.3-fold increase inthe PAH group and a 1.9-fold increase in the PAH+RES groupcompared to CTRL; this represented a 13% decrease in occlu-sion due to the effect of RES when the PAH+RES and PAHgroups were compared (i.e., 30:5 ± 0:9% vs. 26:6 ± 0:9%,respectively) (Figure 1(d)). The rodents treated only withRES showed no differences compared to the CTRL for allthe variables.
These data correlate with the echocardiographic valuespertaining to the pulmonary artery. Compared with theCTRL group, the PAAT decreased significantly in the PAHand PAH+RES groups (i.e., by 40% and 43%, respectively).The PAAT/ET ratio was seen to reduce in correlation with
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Figure 1: There is a limited effect of RES exerted in the lung vessel histopathology structure. (a) Representative microphotographs ofpulmonary blood vessels. PAH induced hypertrophy and proliferation of the tunica media; this effect is decreased by RES. 20xmagnification; H&E staining. Arrows indicate the muscularized vessel wall. (b) Amount of muscular arteries in 7 random fields in lungtissue. (c) Diameter of pulmonary blood vessels. (d) Luminal occlusion by the media layer in lung arteries. The values are given as themean and fold change ± SEM; ∗p < 0:05 vs. control; #p < 0:05 vs. PAH; n = 15 for CTRL, PAH, and PAH+RES; n = 11 for RES.
4 Oxidative Medicine and Cellular Longevity
an increase in pulmonary vascular resistance in both groups,but the change was increased higher in the PAH groupthan in the PAH+RES group (29% and 25%, respectively).The mPAP markedly increased in the PAH and PAH+RES groups (71% and 43%, respectively) compared tothe CTRL group. The mPAP for both groups was estimatedto be higher than 20mmHg, but this change was withoutstatistical significance (Table 1).
3.2. Resveratrol Treatment Improves Right VentricularRemodeling and Function. The values found using TAPSE,a surrogate measurement of right ventricular performance,worsened significantly by 31% in the PAH group comparedto the CTRL group. In contrast with PA hemodynamics, thisvalue improved considerably by 46% in the PAH+RES group,compared to PAH. RV free wall thickness increased markedlyby 60% in the PAH group compared with the CTRL group.However, in the treated PAH+RES group, RV free wall thick-ness decreased significantly by 25% compared with the PAHgroup. The end-diastolic diameter of the RV and the ratio ofthe end-diastolic diameter for the RV/LV increased signifi-cantly in the PAH group compared to the CTRL group. A sig-nificant difference was not observed for both parameters in thePAH+RES group compared to the CTRL group (Table 2). Theeffect of RES, at the microscopic level, on the tissues andisolated cells is depicted in Figure 2(a). To assess tissue remod-
eling, the ventricular wall sections were stained with Masson’strichrome. A multiple-fold increase in fibrosis was observed inthe RV in PAH. RES treatment kept the fibrosis at CTRL levelsin the PAH+RES phenotype, while the effect of RES treatmentalone was the same as that of the CTRL group. The LV in allgroups remained unchanged (Figure 2(d)).
3.3. Resveratrol Restored Right Ventricular CardiomyocyteStructure and Contractile Function in Rodents withPulmonary Arterial Hypertension. The cardiomyocyte areawas analyzed using H&E-stained slides at the level of the pap-illary muscles. The PAH group had this parameter with atleast a two-fold increase (600 ± 23 μm2) compared to theCTRL group (290 ± 11μm2). The cardiomyocyte areadecreased by 17% in the PAH+RES group (498 ± 17:8 μm2)compared to the PAH group (Figure 2(b)). In contrast, a dif-ference between any of the groups was not observed in the LVmyocyte area (Figure 2(d)). The cellular volume of the iso-lated RV cardiomyocytes in rodents with PAH assessed usingconfocal microscopy reflected a 1.7-fold increase compared tothe CTRL group (34:8 ± 1:9 vs. 23:6 ± 1:1 fL, respectively).The PAH+RES group was seen to have a 13% decrease in vol-ume compared to the PAH phenotype (26:8 ± 1:7 fL), andthere was no statistical difference with the CTRL group(Figure 2(c)). There were no differences observed betweenthe LV groups (Figure 2(d)). These changes could indicate
Table 1: Echocardiographic measurements of right ventricle outflow tract flow profiles.
TAPSE: tricuspid annular plane systolic excursion; RV: right ventricle; LV: left ventricle. All data are presented as the mean ± SEM. ∗p < 0:05 vs. CTRL;#p < 0:05 vs. PAH.
5Oxidative Medicine and Cellular Longevity
the prevention or delay of tissue remodeling exerted by RES.Taken together, these data prompted the analysis of func-tional cell shortening in the isolated cells. The effect ofincreased stimulation frequency on cell contractility in theRV and LV cells was evaluated. The cells were paced at 0.5,1.0, and 2.0Hz. The RV CTRL cells had respective values of5:1 ± 2:4%, 4:6 ± 2:4%, and 4:1 ± 1:7%. There were nochanges in the RES-only group compared to the CTRL group(data not shown). In our animal model, the PAH groupexhibited a contractility decrease of 8% at 0.5Hz, 18% at1.0Hz, and 39% at 2.0Hz compared to the CTRL group.Cell shortening in all paces was demonstrated to be sig-nificantly improved in the PAH+RES group comparedto the other groups. Compared to PAH in this regard,there was a respective increase of 59% for 0.5Hz, 71%for 1.0Hz, and 148% for 2.0Hz in the PAH+RES phenotype
(Figure 3(a)). A statistically significant difference was notseen regarding the effect of PAH or RES on the LV cells(Figure 3(b)).
3.4. Inflammatory and Remodeling Effect of RES in RV Tissuefrom Specimens with PAH.We performed qPCR from RV tis-sue to analyze the remodeling and inflammatory markers.We selected BNP, troponin C1 (Tnnc1), and collagen 1 toanalyze the hypertrophy mediated by MC. For the inflamma-tory markers, we chose pro-inflammatory IL-1β and anti-inflammatory IL-10. There was a significant RES-mediateddecrease in the PAH+RES group of the following mRNAscompared to PAH phenotype: BNP (6:7 ± 1:02 vs. 15:7 ±1:5), Tnnc1 (0:7 ± 0:5 vs. 5:5 ± 2:7), collagen 1 (1:7 ± 0:7 vs.2:7 ± 0:5), IL-1β (3:2 ± 0:7 vs. 4:6 ± 1:7), and an increase inIL-10 (8:7 ± 1:5 vs. 3:5 ± 1:6) (Figures 4(a)–4(e)).
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Figure 2: RES reduces the fibrotic index and myocyte hypertrophy in RV of PAH-treated specimens. (a) Fibrotic index. Representative rightventricles stained with Masson’s trichrome of all treated groups (5x). Black arrows indicate the zones of fibrotic tissue. (b) Myocyte area.Representative cross-section cardiomyocytes from groups (H&E, 10x). (c) Cell volume analysis. Representative right ventriclecardiomyocytes stained with calcein and analyzed by confocal microscopy. (d) Unchanged LV morphological features in the PAH modeland the lack of effect of RES on these features. Fibrotic index, myocyte area, and isolated cell volume. All data have been normalized toRV CTRL mean values. The values are given as the mean and fold change ± SEM (n = 15 for CTRL, PAH, and PAH+RES; n = 11 for RES).The values are given as the mean and fold change ± SEM; ∗p < 0:05 vs. CTRL; #p < 0:05 vs PAH.
6 Oxidative Medicine and Cellular Longevity
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Figure 3: RES improves cardiomyocyte shortening isolated from RV, and had with on LV isolated cardiomyocytes. Percentage of cellshortening after 1Hz stimulation in isolated cardiomyocyte from the (a) right ventricle and (b) left ventricle. The values are given as themean ± SEM (∗p < 0:05 vs. control, #p < 0:05 vs. PAH; n = 26-44 cells from 2 animals for CTRL, n = 15-26 cells from 2-3 animals forPAH, and n = 19-61 cells from 2-4 animals for PAH+RES).
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Figure 4: RES modulates the decrease of tissue remodeling and inflammatory mRNA on the RV of PAH-treated specimens. qPCR analysis ofRV from the tissue samples show (a) BNP, (b) troponin C, (c) collagen type 1, (d) IL-1β, and (e) IL-10. All data have been normalized to RVCTRL mean values. The values are given as the mean and fold change ± SEM (n = 4 for CTRL, n = 3 for PAH and PAH+RES, and n = 6 forRES). The values are given as the mean and fold change ± SEM; ∗p < 0:05 vs. control; #p < 0:05 vs. PAH.
7Oxidative Medicine and Cellular Longevity
3.5. There Is a RES-Mediated Increase in SIRT1 in TreatedPAH RV. We found an expected downregulation of SIRT1mRNA in the RV of PAH phenotypic rats compared toCTRL. This decrease (0:5 ± 0:2) was abrogated by thetreatment of RES in these specimens (0:8 ± 0:3)(Figure 5(a)). To evaluate the effect of the decrease inSIRT1 expression, we performed a western blot of theacetylation profile. Treatment of PAH with RES preventedthe increased acetylation of the RV, remaining at the levelof the CTRL group (Figure 5(b)).
4. Discussion and Conclusion
The use of animal models, like the MC-induced PAH in ratshas been an alternative to characterize and explore the phys-iopathology and therapeutics against PAH [19]. The majorityof the literature has focused on relevant topics such aspulmonary changes in the smooth muscle of the vesselsand the incidence of fibrosis [13, 20, 21]. However, it isimportant to highlight the lack of protocols focused onimproving RV function, a critical parameter for outcomeand survival [22]. An example of the clinical relevance ofimproving the ventricular function comes from the revers-ible effects after treating chronic thromboembolic pulmo-nary hypertension (CT-PH), where careful removal of thethrombus reverses the compromised ventricular functionand its remodeling. Therefore, there is a need to continuethe development of coadjuvant treatments for RHF andimprove their mechanisms of delivery [10, 22, 23].
A decision was made to address these issues by studyingthe underlying mechanisms associated with RES treatmenton the cardiovascular and pulmonary system in a valid model
of PAH. The use of RES as an adjuvant in a therapeutic set-ting owing to its multiple effects as a ROS scavenger, mito-chondrial agent, and cell cycle modulator is ongoing andconstitutes promising research [10]. Varying doses of RES[14, 24, 25] and administration routes [25–27] have beenused for numerous therapeutic purposes and diseases. Sev-eral RES doses for PAH models have been described in theliterature, and variable results have been reported [28]. Thecause of this variation relates to the MC dosage, the initialweight of the specimen, and the duration of the study.
For the model in this study, RES at a dose of 20mg/kg/dayby gavage was used, a dose that has been used elsewhere [29].According to the findings of the current study, heart and lungweight increased in both the PAH and PAH+RES groups. Theeffect of RES was not significant, and this phenomenon hasbeen reported in other research [13] and may be associatedwith several factors. An explanation for this is that a higherRES dosage is required for a macroscopic impact.
Consistent and pathognomonic results were found forthe vascular bed of the lungs for all groups treated with thePAH phenotype [30, 31]. RES had a minimal effect in revers-ing them, other authors found that RES had a limited antire-modeling effect on the lungs, and this effect was limited to themedial layer of the pulmonary trunk and did not impact theheart wall structure [13]. In the current study, RES had a par-tial effect on lung histopathology in the PAH+RES-treatedgroup. Using a higher dose and improving the administra-tion route (i.e., using nebulization therapy) could be an effec-tive way of improving these results.
The finding in the current study that RES had a limitedeffect on the lungs correlates with ultrasonographic evidenceof PA. Echocardiography was chosen as it is a noninvasive
CT RL PAH PAH+ RES RES0.0
0.5
1.0
1.5
2.0
mRN
A S
IRT1
/HPR
T
(a)
CTRL PAH PAH+RES
Ac-Lys
GAPDH
C T R L PA H PA H +R E S0 .0
0 .5Ac-
Lys/
GA
PDH
1 .0
1 .5
2 .0
#
(b)
Figure 5: There is a decrease in the mediated SIRT1 deacetylation. (a) qPCR analysis of SIRT1 mRNA expression on heart tissue. (b)Representative western blot membrane of Ac lysine of heart tissue proteins and below the acetylation profile in heart tissue in fold change.For (a), n = 4 for all groups; for (a), n = 3 for all groups. The values are given as the mean and fold change ± SEM; ∗p < 0:05 vs. control;#p < 0:05 vs PAH.
8 Oxidative Medicine and Cellular Longevity
tool that can be used to assess RV function. The current studydemonstrated that it was a feasible technique that could beused in rats for a PAH evaluation, and this has also beendemonstrated by other groups [32]. The PAAT and PAA-T/ET ratio are PA hemodynamic parameters that are highlysusceptible to changes in pulmonary vascular resistance andimpedance [33], and it shortens in correlation with anincrease in systolic PAP and mPAP [34]. Although RES pre-vented the development of specific pathognomonic PAHcharacteristics, it was insufficient to elicit a significant changein surrogate markers of increased mPAP. Chronic RV pres-sure overload has been shown to lead to a gradual changein RV phenotype, which ultimately resulted in RV-arterialuncoupling and subsequent functional deterioration [35]. Asignificantly low value of mPAP was found in the PAH groupin the current research, and more importantly, an improve-ment of this parameter was reported for the PAH+RESgroup, suggesting that treatment with RES prevented systolicfailure, commonly observed in the advanced stages of PAH.Interestingly, this value was even higher in the RES-onlytreatment group, which could indicate that this polyphenolnot only prevents failure but is also a potential enhancer ofRV function [35]. RV free wall thickness, an objective reflec-tion of RV hypertrophy and remodeling [34], was markedlyincreased in rodents with PAH, which is consistent withremodeling secondary to pressure overload. Most impor-tantly, and in contrast with the findings of the PAH group,RES treatment attenuated RV hypertrophy induced by highmPAP, and a significant difference with the CTRL groupwas not observed. This can be secondary to fewer fibroticchanges, which is consistent with previous in vitro researchon the impact of RES on cardiac fibrosis [36]. An increasein the RV end-diastolic diameter was observed in the PAHmodel, and this increase was significant when compared tothe untreated controls, reaching a ratio of ≥1, which is asso-ciated with an increased risk of adverse clinical events, whilebeing a marker for poor prognosis [37]. Dilation and theincreased ratio were not present in the PAH+RES group.Therefore, even with the increased mPAP identified in thePAH+RES group, the RV was able to endure it and preventpathologic remodeling. A further study in this direction isto investigate the effect of RES on cardiac strain because itis possible that enhancement of these variables by RES couldexplain the increased capacity of RV to manage elevated pul-monary pressure. The LV echocardiography findings did notshow any change associated with increased mPAP with REStreatment. Even though its diameter can decrease as a conse-quence of RV dilatation or the presence of pericardial effu-sion, this effect in the PAH groups was not seen uponanalysis. One explanation for the absence of these findingsand for negative ventricular interaction is that the modeldid not allow the development of PAH that was severeenough to cause pericardial effusion or result in higher valuesof mPAP. More research with a focus on echocardiographicchanges is warranted to clarify the changes elicited by MCPAH in LV and LHF.
Signs of increased tissue remodeling consistent withfibrotic changes were identified in the current research aspreviously reported [38, 39]. Interestingly, these changes
focused on the right ventricles of MC-treated rats, while theRES treatment led to a tissue structure that was almost iden-tical to that of the untreated controls. Right heart chambersdiffer from those on the left side, even in terms of theirembryology [40] and primary functions, both physiologicallyand hemodynamically [41].
RES decreased, or, at the very least, inhibited, the pro-gression of fibrosis in the right ventricles of the PAH+RESgroup. This finding suggests that pressure on the heartdecreased after treatment with RES, and this was the primaryreason for validating it using an ultrasonographic assessmentin the current research. RES-mediated molecular mecha-nisms involving TGF-β modulation [42, 43] and its effecton the medial vascular layer have been associated with after-load pressure changes [44]. Other pathways have beendescribed, such as a MC-induced upregulation of SphK1-mediated NF-κB activation, albeit not in the scope of our cur-rent study [45]. Accompanied by a decrease in tissue remod-eling, changes in the myocyte area and isolated cell volumewere found with RES treatment. Consistent with the lack offibrotic changes in the LV, these effects were also absent inthese chambers. The results showed that individual cell vol-ume and the area in the left ventricular cells in all the studiedgroups increased in comparison with those in the healthyCTRL RV cells. Hypertrophic LV compensation has beenreported in heart failure when the MC model was used andis associated with an increase in neurohumoral activation[46]. An interesting finding was that there were no differ-ences between the LV groups (i.e., the PAH and PAH+RESgroups). It could be speculated that remodeling changes inthe LV could appear later. However, this study durationwas twice as long as the typical duration of 21 days. There-fore, this is unlikely. Generally, PAH is characterized by anincrease in pulmonary vascular resistance, which causes RVremodeling and leads to RHF. In the current PAH model,compromised RV function, myocyte hypertrophy, and iso-lated RV cardiomyocytes were identified. Changes in cellularcontractility in the LV were not seen. Interestingly, while adecrease in RV fractional shortening by echocardiography[47] is well known, some studies reported an increase in cel-lular shortening after treatment with MC [48, 49], but thishigher contraction force was not sustained at high-stimulation frequencies [48]; this was also observed in thecurrent study (Figure 3). Consistent with these changes inhypertrophy in an inflammatory model like MC, we foundincreased levels of mRNA of the remodeling markers BNP,collagen 1, Tnn1c, the inflammatory IL-1β, and the anti-inflammatory cytokine IL-10. These markers have beenreported previously for PAH [50, 51], while the cytokineIL-10 has been linked with the increase of fibrosis andTGF-β [52]. RES treatment in PAH decreased the inflamma-tory markers and modulated a decrease of the remodelingeffect (Figure 4). These results also correlate with the effectof RES-mediated SIRT1 upregulation, with a consequentdecrease in the acetylation profile (Figure 5(b)). This hasbeen linked to mitochondrial dysfunction concomitant withventricular dysfunction and heart failure [53].
The increase in myocardial stiffness, as a result of theoverexpression of titin, has been proposed as a possible
9Oxidative Medicine and Cellular Longevity
explanation for contractile dysfunction [54, 55] since thislarge protein is responsible for the passive elasticity of themuscle. Additionally, some changes in calcium handlingcould be involved. For instance, in the PAHmodel, faster cal-cium transients concomitant with increased sarcoplasmicreticulum calcium content and phosphorylation of phos-pholamban were demonstrated. Moreover, calcium sparkfrequency was higher in the RV cardiomyocytes fromrodents in the PAH group. In this regard, RES has been
shown to upregulate the ratio of phosphorylated phospho-lamban and is accompanied by a significant improved sar-coplasmic reticulum calcium load. In the current study,the RES-treated group showed an increase in cellular short-ening; this may have been due to an increase in myocytestiffness as the RV energetic calcium handling might havebeen altered. Impaired mitochondrial function due to hyp-oxia in hypertrophy remodeling has been reported inPAH, which compromises the supply of energy to the
Muscularized arteriesvessel diameter
luminal occlusion
Muscularized arteriesvessel diameter
PV resistancePA pressure
PV resistancePA pressure
RV function: changes inTAPSE RV wall thickness, RVDD and RV/LV
FibrosisMyocyte Areaand CellVolume
FibrosisMyocyte Areaand CellVolume
Cell Shortening
SIRT1
Muscularizedarteries
Alveoli
Normalcardiomyocyte
Fibrosis
Increase
Decrease
SIRT1IL-1𝛽BNP
Troponin
Cell Shortening
RV function: changes inTAPSERV wall thickness, RVDD and RV/LV
in RVAfterload in RV
Afterload
PAH phenotype PAH+RES
IL-1𝛽BNP
Troponin
Figure 6: Effects of resveratrol in the PAH phenotype. Monocrotaline-induced PAH is a disease characterized by a progressive remodeling ofthe pulmonary vasculature, as a consequence of excessive proliferation and migration of pulmonary artery endothelial and smooth musclecells. With the progression of the disease, the increase of the mean pulmonary artery pressure leads to a chamber pressure overload in theright ventricle (RV). When the optimal RV-arterial coupling is lost, the RV systolic function cannot remain matched to the afterload, andsubsequently, dilation of the RV occurs, as well as diastolic dysfunction, secondary to myocardial fibrosis and sarcomeric stiffening. Thesechanges ultimately lead to right heart failure and death. Even though the administration of resveratrol decreased the pathologicalremodeling of the pulmonary vasculature, it did not change the afterload for the RV (represented in the figure as a change in arrows’thickness). Nevertheless, resveratrol was able to protect directly the RV, improving its function, evidentiated with microscopic changes:less fibrosis, decreased cardiomyocyte area and volume and better cell function, with increased cell shortening, increasing SIRT1-mediateddeacetylation, and decreasing inflammatory and remodeling markers. The arrows in the PAH model indicate the changes compared to theCTRL group; the arrows in the PAH+RES model indicate the changes compared to the PAH group.
10 Oxidative Medicine and Cellular Longevity
tissues [56]. Furthermore, creatine kinase activity andexpression have been demonstrated to decrease after MCtreatment [57]. This downregulation has been linked to a com-promise in ATP/ADP transport between the mitochondriaand the myofilaments. RES, besides being a potent antioxi-dant, is known for its cardioprotective action as it preservesthe mitochondrial function by regulating the activity of anti-oxidant enzymes by reducing ROS [58].
PAH remains a challenging disease owing to its highmorbidity, mortality, and time to diagnose. Although currenttherapies are improving the quality of life and survival, thereis a need to identify adjunctive treatments focussing on RVfunction. As a result, PAH continues to be revisited, and anincreasing number of therapeutic approaches are evaluatedannually. However, the current guidelines for pharmacother-apy primarily focus on the vascular effects [59]. Since the dis-ease is usually diagnosed late, a large amount of structuraldamage in the RV has already taken place. Contrary to somemodels where RES has been reported to have an effect on thepulmonary trunk [13] and although using an inflammatorymodel such as MC, the findings of the current study demon-strated that RES improved the RV and had a limited positiveeffect on the lungs (Figure 6). This finding is crucial since RVfunction correlates with symptomatology and the prognosticsurvival of the patients. Focusing on comparative ventricularassessment and isolated cell function, the current studyshowed how RES-mediated mechanisms might be involvedusing this model. Some other mechanisms include activationof specific sirtuin pathways like SIRT3, the modulation ofcardiac energetics [53], and transcription factors relating toproliferation and the cell cycle [11]. Possible novel targetsthat focus on RV HF could become an exciting future scopefor therapy. The multitargeted nature of RES, as an exampleof these polyphenolic compounds, holds future potential fornovel approaches to this disease [60]. Ongoing research isneeded to help characterize the molecular mechanisms andcell bioenergetics of these compounds in PAH and othercardiovascular conditions.
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request.
Conflicts of Interest
The authors declare that there is no conflict of interestregarding the publication of this paper.
Acknowledgments
This work was partially supported by the Cardiovascu-lar Medicine Research Group-Tecnológico de Monterrey(0020CAT131), CONACYT-México (grants 151136, 256577,and A1-S-43883 (G. García-Rivas), A1-S-23901 (OmarLozano), 492122 (Judith Bernal)), and Fronteras de la Ciencia(Grant 0682). We thank Paulo Martinez, DVM, for hisexceptional veterinarian assistance.
Supplementary Materials
Supplementary Table 1: primer sequences for real-timeqPCR in heart tissue. (Supplementary Materials)
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